This application is a 371 of PCT/JP97/02985, filed Aug. 27, 1997.
The present invention relates to a novel hematopoietic growth factor protein termed hematopoietic stem cell growth factor (hereinafter referred to as xe2x80x9cSCGFxe2x80x9d) which acts on hematopoietic stem cells to maintain their survival and to induce their proliferation and differentiation. The present invention also relates to a gene coding for SCGF; a vector comprising the gene; a transformant transformed with the vector; a method for producing SCGF; and a method for separating and purifying SCGF. The present invention further relates to the use of SCGF as a therapeutic for hematopoietic insufficiency derived from irradiation or chemotherapy for patients with various hematopoietic diseases or cancers; or the use of SCGF as a reagent for diagnostic analysis. The present invention also relates to the use of SCGF in bone marrow transplantation for the purpose of hematopoietic recovery, for which hematopoietic stem cells can be amplified with SCGF in vitro in a small amount of bone marrow aspirates; and the use of SCGF in gene therapy to improve the efficiency of a gene transfer into hematopoietic stem cells. The invention also relates to a vector developed to isolate the SCGF gene, as well as a method for isolating the gene. The vector and the method can provide promising tools to search for other novel genes for unknown proteins.
Hematopoiesis in the bone marrow is regulated by direct interaction between (i) self-renewable hematopoietic stem cells, hematopoietic progenitor cells derived therefrom and committed to respective differentiation pathways and cell populations at consecutive differentiation stages between the above two types of cells, and (ii) stromal cells as hematopoietic inductive microenvironment supporting the above cells, or by indirect interaction between (i) cells and hematopoietic humoral factors secreted by (ii) cells. A number of hematopoietic humoral factors are also secreted by extramedullary organs such as the kidney or the liver. Peripheral blood cells with a limited life span are continuously recruited through the hematopoietic network spreading over the whole body which results in maintaining the hamarological stasis. The complicated hematopoietic mechanisms have been analyzed using the following two approaches; first, the process of hematopoietic recovery from myelosuppression is studied in vivo in the experimental animals such as mouse, dog or sheep, which are irradiated or given cytotoxic reagents such as 5-fluorouracil. Second, interaction between hematopoietic stem cells and stromal cells or humoral factors is studied in vitro, using a clonal culture of the human and mammalian bone marrow cells.
With the progress in molecular biology, a number of genes for cytokines including hematopoietic growth factors have been successfully cloned. Such cytokines include erythropoietin (hereinafter referred to as xe2x80x9cEpoxe2x80x9d), thrombopoietin, colony stimulating factors such as granulocyte colony-stimulating factor (hereinafter referred to as xe2x80x9cG-CSFxe2x80x9d), macrophage colony-stimulating factor (hereinafter referred to as xe2x80x9cM-CSFxe2x80x9d), granulocyte macrophage colony-stimulating factor (hereinafter referred to as xe2x80x9cGM-CSFxe2x80x9d) interleukins such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-9, IL-11 and IL-12 recently identified stem cell factor (SCF) and flk-2/flt3 ligand (Lin et al., Proc. Natl. Acad. Sci. USA 82, 7580-7584, 1985; de Sauvage et al., Nature 369, 533-538, 1994; Nagata et al., EMBO J. 5, 575-581, 1986; Wong et al., Science 235, 1504-1508, 1987; Miyatake et al., EMBO J. 4, 2561-2568, 1985; Clark et al., Nucleic Acids Res. 14, 7897-7914, 1986; Dorssers et al., Gene 55, 115-124, 1987; Yokota et al., Proc. Natl. Acad. Sci. USA 83, 5894-5898, 1986; Campbell et al., Proc. Natl. Acad. Sci. USA 84, 6629-6633, 1987; Yasukawa et al., EMBO J. 6, 2939-2945, 1987; Yang et al., Blood 74, 1880-1884, 1989; Paul et al., Proc. Natl. Acad. Sci. USA 87, 7512-7516, 1990; Wolf et al., J. Immunol, 146, 3074-3081, 1991; Anderson et al., Cell 63, 235-243, 1990; Lyman et al., Cell 75, 1157-1167, 1993). These cytokines have been well characterized biologically and biochemically, and their industrial production has become possible. G-CSF, M-CSF and Epo are used clinically as recombinant preparations for hematopoietic insufficiency derived from irradiation or chemotherapy and anemia associated with renal failure, respectively. However, when hematopoietic insufficiency due to quantitative or qualitative hematopoietic stem cell abnormalities is treated with the recombinant hematopoietic growth factors, peripheral blood counts are only transiently improved. Hematopoietic insufficiency often recurs with cessation of the hematopoietic growth factors. In other words, presently available hematopoietic growth factors have not achieved radical cure of hematopoietic insufficiency due to hematopoietic stem cell abnormalities.
Auto- or allo-graft of bone marrow, peripheral blood and cord blood hematopoietic stem cells are common procedures for hematopoietic insufficiency. On the other hand, hematopoietic stem cells in the bone marrow, peripheral blood or cord blood cells are tried to be the amplified in vitro with hematopoietic growth factors and then transplanted. Among the factors described above, SCF, IL-3, G-CSF and IL-6 play a major role in amplification of hematopoietic stem cells and immature progenitors. These factors are known to exhibit the so-called xe2x80x9csynergistic effectxe2x80x9d, i.e. they induce higher amplification when used in combination than when used alone. Mouse hematopoietic stem cells can be amplified in vitro to 10-fold and progenitor cells to 1000-fold in response to the hematopoietic growth factors. In human, however, an expected amplification effect has not been achieved with the combination of SCF, IL-3, G-CSF and IL-6 that is effective in the mouse system (Bernstein et al., Blood 77, 2316-2321, 1991; Brandt et al., Blood 79, 634-641, 1992; Srour et al., Blood 81, 661-669, 1993). This not only implies that human cells expressing the receptors for the hematopoietic growth factors are different from mouse ones, but strongly suggests the existence of unknown factors involved in human hematopoiesis.
When host cells are transfected or infected, in gene therapy, with a retrovirus vector carrying a normal gene or a gene of interest, the efficiency of gene transfer will be extremely low if the host cells are not in the cell cycle and, as a result, no expression of the gene can be achieved. If a gene is transferred into the short-lived mature blood cells, gene therapy should be repeated many times since the expression of the gene is transient. Therefore, hematopoietic stem cells are a preferable target for gene transfer, for the reason that it is therapeutically excellent to transfer a gene of interest into hematopoietic stem cells once to thereby supply cells expressing the gene permanently. However, since hematopoietic stem cells are usually quiescent in the G0 phase, attempts have been made to enter them into the cell cycle using a combination of hematopoietic growth factors such as SCF, IL-3, G-CSF, IL-6 and so forth. The efficiency of gene-transfer is still as low as 40%, which is the biggest problem in gene therapy (Nolta et al., Hum. Gene Therapy 1, 257-268, 1990; Stoeckert et al., Exp. Hematol. 18, 1164-1170, 1990; Dick et al., Blood 78, 624-634, 1991; Cournoyer et al., Hum. Gene Therapy 2, 203-213, 1991; Hughes et al., J. Clin. Invest. 89, 1817-1824, 1992).
Hiraoka et al. have found a growth activity on human hematopoietic stem cells in the culture supernatant of normal human peripheral blood mononuclear cells and that of undifferentiated myeloid KPB-M15 cells established from the peripheral blood leukocytes of the patient with chronic myelogenouse leukemia in blast crisis, designated the activity xe2x80x9chematopoietic stem cell growth factorxe2x80x9d (SCGF) and tried to purify the factor (Hiraoka et al., Cell Biol. Int. Rep.10, 347-355, 1986; Hiraoka et al., Cancer Res. 47, 5025-5030, 1987). The hematopoietic activities of SCGF include, erythroid burst-promoting activity (hereinafter referred to as xe2x80x9cBPAxe2x80x9d) in the presence of Epo and granulocyte macrophage colony-promoting activity (hereinafter referred to as xe2x80x9cGPAxe2x80x9d) in the presence of GM-CSF on human bone marrow cells, while SCGF lacks colony-stimulating activity (hereinafter referred to as xe2x80x9cCSAxe2x80x9d).
Since human SCGF shows a strict species-specificity, i.e. it is active on human hematopoietic stem or progenitor cells but not on mouse cells, investigators should not use mouse bone marrow cells but human cells for purification and identification of SCGF. Human bone marrow cells are least available due to the limited number of donors, so the progress in the studies is limited and it has been remained ambiguous whether SCGF is different from or identical with a known factor.
It is an object of the invention to identify the molecular characteristics of the novel hematopoietic growth factor xe2x80x9cSCGFxe2x80x9d through purification of SCGF protein and cloning of a gene coding for SCGF, and to provide the recombinant SCGF preparation. The present invention intends to contribute to the in vitro amplification of human hematopoietic stem and progenitor cells, amelioration of various hematopoietic disorders, gene therapy and the diagnosis of diseases. In view of the species-specificity of SCGF, the present invention intends to make clear an important aspect of SCGF-concerned hematopoietic mechanisms unidentified expanded from the studies using mouse bone marrrow cells.
Since recombinant DNA technology has remarkably advanced, isolation and identification of a gene has become possible even for quite a small amount of a physiologically active protein (Huynh et al., DNA Cloning I, A Practical Approach, Glover (ed.), Oxford, Wash., IRL Press, 49-78, 1985; Sambrook, Fritsch and Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Springs Harbor Laboratory Press, 1989). There are two major methods for isolating a gene. First is a protein-purification leading method, to determination of the partial amino acid sequence; an oprimal oligo-DNA probe is prepared based on the amino acid sequence, then a relevant gene is screened to hybridize with the DNA probe (i.e., a gene which is expected to encode the same amino acid sequence as that of the purified protein), from a large-scale cDNA library. Second is an expression cloning method; a protein is secreted by cells expressing a relevant cDNA clone and detected using an activity assay system or a specific antibody. The latter is superior to the former in that the protein need not be highly purified for isolation of the gene. It means the expression cloning method requires the specific antibody or sensitive activity assay system for successful gene isolation.
The present inventors have modified and improved the expression cloning method. First, the genes totally different from that for SCGF have been tried to be excluded from a cDNA library. Briefly, the positive DNA probes was prepared from DNA of SCGF-producing KPB-M15 cells subtracted with SCGF-infertile MOLT-4 cells, and the negative probe from DNA of the MOLT-4 cells. For a differential cloning, the genes that hybridize with the positive probe but not with the negative probe were sorted from a cDNA library of KPB-M15 cells. Somewhat less sensitive assay system for SCGF activity can be appropriate to detect SCGF cDNA clone by reducing the number of expression samples from the sorted cDNA clones. While a cDNA should be ligated to a phage vector for differential hybridization, it be ligated to a plasmid vector for expression of the cDNA in mammalian cells to rest the SCGF activity of the protein. In order to meet the demand, the inventors have developed a novel vector convertible from a phage vector to a plasmid one on the same vector. Briefly, genes for replication in E. coli and expression in mammalian cells were ligated into a xcex phage vector with a flanking replication initiator and terminator. The xcex phage vector was packaged and coinfected with a helper phage into E. coli, replicating to circularize the region between the initiator and terminator. The replicated circular DNA was covered with the coat protein to develop an infectious phagemid. When E. coli was infected with, the phagemid, the circular DNA in the phagemid was transferred into the E. coli, resulting in transformation of E. coli. The DNA between replication initiator and terminator in the xcex phage vector was consequently transferred into E. coli to be plasmid-like. Since the plasmid-like circular DNA had the ability to replicate in E. coli and to be expressed in mammalian cells, the initial xcex phage vector had been converted to a plasmid vector without DNA recombination.
A KPB-M15 cDNA library was prepared into the above vector. About 60,000 cDNA clones were sorted to about 6,800 through differential cloning. Gene products expressed in COS cells were screened for BPA. cDNA clone No. 116-10C was isolated as that for SCGF. Nucleotide sequencing showed that the cDNA had 1,196 nucleotides with a long open reading frame that encoded a 245-amino acid polypeptide. About 20 amino acids at an N-terminal region were hydrophobic. No homology with the database in the EMBL and GenBank was found for the cDNA sequence in the total or most of the coding region, though only one short DNA fragment showed partially high homology with the 3xe2x80x2 untranslated region of the cDNA clone No. 116-10C. Furthermore, no homology with database in the Swiss-Prot was found for the amino acid sequence. Collectively, cDNA clone No. 116-10C has been confirmed to be a gene coding for the novel hematopoietic growth factor SCGF. Thus, the present invention has been achieved.
Hereinbelow, the present invention will be described in detail.
First, the invention relates to a mammalian polypeptide with BPA or GPA on the bone marrow cells.
Secondly, the invention relates to a mammalian gene that encodes a polypeptide with BPA or GPA on the bone marrow cells.
Thirdly, the invention relates to a vector carrying the above gene.
Fourthly, the invention relates to a transformant transformed with the above vector.
Fifthly, the invention relates to a specific antibody for the above polypeptide.
Sixthly, the invention relates to microorganisms, animal cells or animals producing the above antibody.
Seventhly, the invention relates to a method for producing the above polypeptide by the culture cells possessing the above gene.
Eighthly, the invention relates to a method for purification of the above polypeptide using one or more of an anion exchange, a hydrophobic, a gel filtration, a pigment and lectin affinity, and a metal-chelating chromatography.
Ninthly, the invention relates to a pharmaceutical composition the above polypeptide as an active ingredient.
Tenthly, the invention relates to a xcex phage vector which has at least 2 functional DNA regions for replication in E. coli and for expression in mammalian cells with a flanking replication initiator and a terminator from a filamentous phage.
Eleventhly, the invention relates to a method for isolating a gene using the above xcex phage vector.
(1) The Polypeptide of the Invention (the 1st Invention)
The polypeptide of the invention is that of a mammalian origin possessing BPA or GPA on the bone marrow cells.
BPA can be detected by erythroid bursts formation in a soft agar culture of the bone marrow cells in response to Epo and the polypeptide to be tested. For example, bone marrow cells are seeded at a density of 5xc3x97104 /ml into 0.3% agar medium, containing 1 unit/ml of Epo and the polypeptide. Erythroid bursts consisting of erythroblasts are enumerated under an inverted microscope after 14-day culture. GPA can be detected by an increase in the number of GM colonies formed in a soft agar culture of the bone marrow cells in response to GM-CSF and the polypeptide. For example, bone marrow cells are seeded at a density 5xc3x97104 /ml in 0.3% agar medium, containing 5 ng/ml of GM-CSF and the polypeptide. GM colonies consisting of granulocytes and macrophages are enumerated under an inverted microscope after 10-day culture. Bone marrow cells are aspirated from the mammalian sternum or ilium, and suspended in a medium, e.g. Iscove""s modified Dulbecco""s medium (IMDM) containing 10% fetal calf serum (FCS). Low density mononuclear cells are separated by centrifugation on a high density isotonic cushion, e.g. Ficoll.
The polypeptide of the invention can be obtained by the cultures of cells possessing the gene of the invention (the 2nd invention) described below.
Human placenta and KPB-M15 cells are candidates of cells possessing the gene of the invention. Cells with the recombinant gene of the invention are another sources.
Structure of the purified polypeptide of the invention can be analyzed by conventional methods used in protein chemistry, e.g. the method described in Hisashi Hirano, Structural Analysis of Proteins for Gene Cloning, Tokyo Kagaku Dojin Co., 1993.
In addition to the use as a pharmaceutical composition described below, the polypeptide of the invention can be used for auto- or allograft of bone marrow, peripheral blood and cord blood hematopoietic stem cells. Hematopoietic stem cells in the bone marrow, peripheral blood or cord blood cells are amplified in vitro with the polypeptide of the invention alone or in combination with hematopoietic growth factors such as G-CSF, GM-CSF, SCF, flk-2/flt3 ligand, IL-1, IL-3 and IL-6. Therefore, bone marrow cells need not be aspirated in an operating room, but a small amount of bone cells should be aspirated easily in a short time at an outpatient clinic which will be sufficient for transplantation. The physical burden of a cell donner, the labor of a medical staff, and the medical cost, associated with bone marrow aspiration, can be saved.
Among the polypeptides of the invention, the following 4 polypeptides described below are particularly preferred.
(i) The following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on human bone marrow cells.
(ii) The following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on human bone marrow cells.
(iii) The following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on mouse bone marrow cells.
(iv) The following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on rat bone marrow cells.
The xe2x80x9cdeletion, substitution or additionxe2x80x9d mentioned herein can be generated by conventional techniques at the time of the filing of this application, e.g. site-specific mutagenesis (Zoller et al., Nucleic Acids Res. 10, 6487-6500, 1982).
KPB-M15 cell line capable of producing the polypeptide (i) above has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba City, Ibaraki Pref., Japan) under Accession No. FERM BP-5850 (date of deposit: Mar. 5, 1997).
(2) The Gene of the Invention (the 2nd Invention)
The gene of the invention is that of a mammalian origin encoding a polypeptide with BPA or GPA on the bone marrow cells.
BPA and GPA can be detected as described above.
The gene of the invention can be synthesized from mRNA by PCR on the cDNA from mammalian mRNA as a template, using a forward and a reverse primers synthesized based on the nucleotide sequence shown in SEQ ID NO: 2. Specific examples of the mammals to be used in the invention include, but are not limited to, human and mouse. Specific examples of the primers for PCR include, but are not limited to, the primer shown in SEQ ID NO: 6 and the primer shown in SEQ ID NO: 7. mRNA preparation, cDNA synthesis and PCR can be carried out by conventional methods.
The gene of the invention will be useful as a template essential for a large scale production of a recombinant mammalian SCGF using recombinant DNA technology. SCGF-producing cells can be identified by in situ or Northern hybridization with a part of the DNA sequence of the gene. The genomic DNA for SCGF can be simiarly isolated and characterized. The present gene of the invention can contribute to the diagnosis of various hematopoietic diseases or elucidation of the pathogenesis by analysis for deletion, mutation, and suppressed or excessive expression of the gene. The present invention is accordingly applicable to gene therapy, e.g. introduction of a delered gene, replacement of a mutated gene with a normal one, suppression of excessive gene expression with an antisense DNA (RNA), and so on.
It is possible to isolate and characterize other mammalian SCGF genes highly homologous with human and mouse ones, using a part of the gene of the invention as a probe. Knock-out and transgenic animals constitutively lacking and expressing SCGF gene respectively, are quite significant in analysis for pathogenesism disease model animals and SCGF-concerned hematopoietic mechanism per se.
Among the genes of the invention, the following 4 genes are particularly preferred.
(i) A gene encoding the following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on human bone marrow cells.
(ii) A gene encoding the following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on human bone marrow cells.
(iii) A gene encoding the following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on mouse bone marrow cells.
(iv) A gene encoding the following polypeptide (a) or (b):
(a) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12;
(b) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 12 with deletion, substitution or addition of one or more amino acids and having BPA or GPA on rat bone marrow cells.
The xe2x80x9cdeletion, substitution or additionxe2x80x9d can be generated by site-specific mutagenesis, as described above.
An E. coli carrying the gene described in (i) above (Escherichia coli SHDM11610C) has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-chome, Tsukuba City, Ibaraki Pref., Japan) under Accession No. FERM BP-5849 (date of deposit: Mar. 4, 1997); an E. coli carrying the gene described in (ii) above (Escherichia coli HSCGF) has been deposited with the same institute under Accession No. FERM BP-5986 (date of deposit: Jun. 19, 1997); an E. coli carrying the gene described in (iii) above (Escherichia coli MSCGF) has been deposited with the same institute under Accession No. FERM BP-5987 (date of deposit: Jun. 19, 1997); and an E. coli carrying the gene described in (iv) above (Escherichia coli RSCGF) has been deposited with the same institute under Accession No. FERM BP-6063 (date of deposit: Aug. 19, 1997).
(3) The Vector of the Invention (the 3rd Invention)
The vector of the invention carries the gene of the invention described above. The vector may have additional DNA region, e.g. a replication initiator and terminator, a selective marker gene(s), a promoter to enhance the expression of the gene of the invention, a poly(A) (polyadenylation) signal, and so forth.
The vector of the invention can be prepared by inserting the gene of the invention into the restriction site of a known vector, such as plasmid, cosmid, phage or virus, digested with an appropriate restriction enzyme. Specific examples of the known vectors to be used in the invention include, but are not limited to, pBR322, PACYC, pUC, PGEM, pBC, pGA, Bluescript, pK21, pRSV, pcD, pGEX, CDM8, SHDM, pBV, pSV, pMT2, pYAC, pWE15, pHEBo, EMBL, Charon, M13, xcex zap, xcex SHDM, xcex gt10 and xcex gt11 vectors.
(4) The Transformant of the Invention (the 4th Invention)
The transformant of the invention is transformed with the vector of the invention described above. The transformant of the invention can be from any organism species if transformed with the above vector.
The transformant of the invention can be prepared by transforming an appropriate host with the above vector. E. coli, yeast, insect and mammalian cells are candidates of the host to be used in the invention. More specifically, the hosts include, but are not limited to, E. coli strains such as HB101, JM109, MC1061, BL21, XL1-Blue, SURE, DH1, DH5; yeast strains such as HIS/LI, HF7c; insect cells such as BmN, Sf cells; and mammalian cells such as CHO, COS, MOP, c127, Jurkat, WOP, HeLa, Namalwa cells. An appropriate method of transformation should be selected depending on the host; the calcium phosphate precipitation or electroporation for E. coli; the lithium acetate method, spheroplast fusion or electroporation for yeast; viral infection for insect cells; the calcium phosphate precipitation, protoplast fusion, lipofection, the erythrocyte ghost method, liposome fusion, the DEAE-dextran method, electroporation or viral infection for mammalian cells.
(5) The Antibody of the Invention (the 5th Invention)
The antibody of the invention reacts specifically with the polypeptide of the invention described above. The antibody of the invention can be either a monoclonal or a polyclonal antibody if specifically reactive with the above polypeptide.
The antibody of the invention can be prepared by conventional methods. In vivo method and in vitro method are a single or multiple immunization, at several week interval, with antigen and immunocompetent cells plus adjuvant, respectively. Specific examples of the immunocompetent cells capable of producing the antibody of the invention include spleen, tonsil and lymph node cells. Whole molecule of the polypeptide of the invention need not be necessarily used as an antigen, but a part of the polypeptide may well be antigenic. A short polypeptide, particularly of about 20 amino acids, should be chemically linked to a highly antigenic carrier protein such as Keyhole Lympet hemocyanin or bovine serum albumin. Alternatively, the polypeptide is covalently bound to a branched skelton polypeptide such as lysine-core MAP (Posnett et al., J. Biol. Chem. 263, 1719-1725, 1988; Lu et al., Mol. Immunol. 28, 623-630, 1991; Briand et al., J. Immunol. Methods 156, 255-265, 1992). Complete or incomplete Freund""s adjuvant and aluminium hydroxide gel can be used as the adjuvant. Animals to be immunized with the antigen include mouse, rat, rabbit, sheep, goat, chicken, cow, horse, guinea pig and so forth. Blood is obtained from animals immunized, and polyclonal antibody immunoglobulin is purified from serum by ammonium sulfate precipitation, anion exchange, protein A or G chromatography. An antibody can be purified from eggs in the case of immunized chicken. Immunocompetent cells (immunized in vitro or recovered from the immunized animals as described above) are fused with parental cells, giving rise to hybridoma cells. Monoclonal antibody is purified from culture supernatants of the hybridoma cells or ascites of animals transplanted with the hybridoma cells intraperitoneally. Specific examples of the parental cells include X63, NS-1, P3U1, X63.653, SP2/0, Y3, SK0-007, GM1500, UC729-6, HM2.0 and NP4-1 cells. Alternatively, immunocompetent cells (immunized in vitro or recovered from the immunized animals) are infected with appropriate virus such as EB virus, resulting in immortalized, antibody-producing cells, from which monoclonal antibody can be prepared. Genetic engineering can be applied to antibody production. For example, an antibody gene can be amplified by PCR from immunocompetent cells (immunized in vitro or recovered from the immunized animals), and transferred into E. coli to produce the antibody. Alternatively, the antibody can be expressed as a fusion protein on the surface of a phage.
immunoassay for SCGF concentration in patients tissues or organs, using the antibody of the invention, elucidates relationship between SCGF and pathogenesis or clinical course of various diseases. The antibody of the invention is versatile for diagnosis, treatment of diseases and efficient affinity purification of SCGF.
(6) The Antibody-Producing Organism of the Invention (the 6th Invention)
The microorganisms, animal cells or animals of the invention produce the antibody of the invention described above. Specific examples of the microorganisms, animal cells or animals include, but are not limited to, E. coli transformed with the gene encoding the antibody of the invention; a phage expressing the gene for antibody of the invention as a fusion protein on the surface, and the hybridoma cells described above.
(7) Method for Producing the Polypeptide of the Invention (the 7th Invention)
The polypeptide of the invention can be produced by the culture of cells carrying the gene of the invention described above.
There exist two types of cells that express the gene of the invention; human placenta and KPB-M15 cells naturally express the gene, and COS and CHO cells should be transferred with the gene. The polypeptide of the invention can be purified from the culture supernatants of the above cells using purification method of the invention described below. Alternatively, the polypeptide can be extracted from cellular lysates. The cells are broken by physical shearing using a clounce homogenizer or ultrasonication, or lyzed with surfactants such as Triton X-100, Nonidet P-40 and sodium lauryl sulfate (SDS).
(8) Method to Purify the Polypeptide of the Invention (the 8th Invention)
The polypeptide of the invention can be purified using one or more of an anion exchange, a hydrophobic, a gel filtration, a pigment and lectin affinity, and a metal-chelating chromatography.
Crude sample is applied to an anion exchange, for example, DEAE-Sephacel column, washed to let unabsorbed proteins flow-through, and eluted with a linear increasing NaCl gradients. Crude sample is applied to a hydrophobic, for example, Octyl-Sepharose column, and flow-through fractions are collected. Crude sample is fractionated through a gel filtration, for example, Sephacryl S-200 HR column. Crude sample is applied to a pigment affinity, for example, Red- or Blue-Sepharose column, and flow-through and early eluted fractions are collected. Crude sample is applied to a lectin affinity carrier, for example, wheat germ agglutinin (WGA)-agarose or Concanavalin A (ConA)-Sepharose column, and flow-through fractions are collected. Crude sample is applied to a metal-chelating, for example, Cu2+-chelating Sepharose column, washed to let unadsorbed molecules flow-through, and eluted with a linear increasing glycine gradients.
(9) The Pharmaceutical Composition of the Invention (the 9th Invention)
The pharmaceutical composition of the invention comprises the polypeptide of the invention as an active ingredient.
The pharmaceutical composition of the invention is administered alone or in combination with other hematopoietic growth factor(s) systemically or locally, and orally or parenterally. The composition is administered at an effective close to ameliorate hematopoietic insufficiency by the hematopoietic activity of SCGF. However, the dosage should be flexible and proper, since the administration dose varies depending on the age, body weight, conditions and reactivity of a patient, route of administration and so on.
For oral administration, the pharmaceutical composition of the invention can be as a solid composition such as tablets, soft and hard capsules, pills, powder and granules, or a liquid composition such as solution, syrup and suspension. For parental administration, the pharmaceutical composition of the invention can be an endermic formulation for external use such as solution, suspension, emulsion, ointment, cream, gel, rosol; a suppository; or an injectable formulation for intravenous, intramuscular or subcutaneous injection.
A pharmaceutically inactive carrier for the pharmaceutical composition can be a diluent such as purified water, lactose, glucose, starch, mannitol, hydroxypropylcellulose, polyvinylpyrrolidone, magnesium aluminate metasilicate, gum-arabic, talc, a vegetable oil or yellow petrolatum. Further, pharmaceutically active additives can be any of conventional, pharmaceutically acceptable materials such as lubricants (magnesium stearate, etc.), disintegrators (fibrin calcium glycolate, etc.), stabilizers (human serum albumin, etc.) and resolution adjuncts (arginine, aspartic acid, etc.).
The solid composition for oral administration can be tablets formulation coated with one or more layers of white sugar, gelatin, hydroxypropyl-cellulose and hydroxypropylmethylcellulose phthalate to be dissolved in the stomach or small intestine. The liquid composition for oral administration can contain purified water, ethanol, solutions, syrups, suspensions, emulsions, elixirs and so on. Further, the composition can contain flavoring agents, preservatives and the like in accordance with appropriate standards for pharmaceutical combination.
The injectable formulation for intravenous, intramuscular or subcutaneous injection can contain, as a pharmaceutically inactive carrier, a diluent such as distilled water for injection, physiological saline, propyrene glycol, polyethylene glycol, a vegetable oil (e.g., olive oil) or an alcohol (e.g., ethanol). In addition, the formulation can contain, as pharmaceutically active additives, preservatives, stabilizers, emulsifiers, buffers, dispersants, resolution adjuncts and the like in accordance with appropriate standards for pharmaceutical combination. The injectable formulations are sterilized by filter-sterilization, addition of a bactericide, or irradiation. Alternatively, a solid composition (such as a lyophilized preparation) can be restored with an aseptic distilled water for injection or the like before administration.
The pharmaceutical composition of the invention is used alone or in combination with other hematopoietic growth factor(s) (such as Epo, G-CSF, GM-CSF and SC) to ameliorate such hematopoietic insufficiency that could not have been improved with known hematopoietic growth factors. Hematopoietic insufficiency to be treated with the pharmaceutical composition of the invention include hematopoietic diseases due to quantitative or qualitative abnormalities in hematopoietic stem cells; aplastic anemia, paroxysmal nocturnal hemoglobinurea, chronic myelocytic leukemia, polycythemia vera, essential thrombocythemia, myelofibrosis, myelodysplastic syndrome and acute leukemia; and hematological diseases such as megaloblastic anemia, AIDS, multiple myeloma, metastatic cancer of the bone marrow, and drug-induced myelosuppression. The pharmaceutical composition of the invention is effective to prevent and ameliorate hematopoietic insufficiency due to irradiation or chemotherapy of the patients with malignant lymphoma or other solid tumors.
The pharmaceutical composition of the invention is applicable to gene therapy; supplementation of a normal gene into enzyme deficiencies (e.g. adenosine deaminase deficiency); replacement of an abnormal gene with a normal gene in genetic mutations (e.g. hemoglobin opathy); and introduction of a gene encoding growth inhibitory factors against cancer cells. Since quiescent hematopoietic ste, cells can enter the cell cycle with the composition of the invention alone or in combination with other hematopoietic growth factor(s) transfection efficiency of a retrovirus vector carrying a normal gene or a gene of interest into those cells is remarkably improved. Once hematopoietic stem cells into which the gene has been introduced are transplanted, they continuously recruit mature blood cells carrying the gene to alleviate or cure the relevant genetic disorders.
(10) The xcex Phage Vector of the Invention (the 10th Invention)
The xcex phage vector of the invention has at least 2 functional DNA regions for replication in E. coli and for expression in mammalian cells with a flanking replication initiator and a terminator from a filamentous phage.
A filamentous phage is a bacteriophage with a single-stranded circular DNA, which specifically infects F fractor-containing E. coli. Specific examples of the phage include M13, f1 and fd phages. xe2x80x9cA replication initiatorxe2x80x9d is a nucleotide region which the filamentous phage recognizes to start DNA replication e.g. the nucleotide base sequence of the NheI-DraIII region of M13 phage replication origin gene (ori). xe2x80x9cA replication terminatorxe2x80x9d is a nucleotide region which the filamentous phage recognizes to stop DNA replication, e.g. the nucleotide sequence of the AvaI-RsaI region of M13 phage ori gene. The xcex phage vector of the invention should have at least 2 functional DNA regions for replication in E. coli and for expression in mammalian cells with a flanking replication initiator and a terminator of a filamentous phage. xe2x80x9cA DNA region for replication in E. colixe2x80x9d is a replication initiation region (ori) of E. coli, e.g. the ColE1 ori. xe2x80x9cA DNA region for expression in a mammalian cellsxe2x80x9d consists of at least a promoter and a poly(A) addition signal for efficient expression of a foreign gene based on DNA from a virus capable of infecting mammalian cells. A virus capable of infecting mammalian cells include SV40 virus, BK virus, papilloma virus, adenovirus, retrovirus, vaccinia virus, EB virus and so forth. A xcex phage vector is a vector from bacteriophage xcex-derived DNA. Specific examples of xcex phage vector include xcex SHDM and xcex CDM both described in Examples of the present invention, xcex gt10, xcex gt11, EMBL3, EMBL4 and Charon4A.
The vector of the invention and the method of the invention for isolating a gene described below are applicable to the search for other novel genes, and can contribute to the technical development in novel genes-related the fields of genetic engineering and biotechnology.
(11) The Method of the Invention for Isolating a Gene (the 11th Invention)
The method of the invention for isolating a gene utilizes the xcex phage vector of the invention described above. Specifically, a method consisting of the following 4 steps may be illustrated by example, but the method of the invention is not limited to this method.
A) First Step
mRNA is prepared from cells producing a protein of interest. cDNA is synthesized from the mRNA, and ligated into the cloning site between the replication initiator and terminator of the xcex phage vector of the invention. A host cell is infected with the recombinant phage vector to provide a phage cDNA library. A protein of interest can be, but is not limited to, the polypeptide of the invention (SCGF). Any protein can be used if it has some activity or function assayable for screening. Cells producing a protein of interest can be, but are not limited to, KPB-M15 cells producing SCGF. Alternatively, a protein of interest can be detected with the specific antibody. mRNA can be prepared, and cDNA synthesized using conventional methods. Host cells to be infected can be conventional host cells such as E. coli. 
B) Second Step
The specific differential phage cDNA library is made of clones that hybridize with a positive probe but not with a negative probe.
The positive probe is single-stranded cDNA (sscDNA) synthesized from the mRNA of the cells producing the protein of interest and subtracted with the mRNA of cells that closely resemble the above cells but do not produce the protein of interest. The negative probe is an sscDNA synthesized from the mRNA of the cells not producing the protein of interest. Differential cloning with the positive and the negative probes exclude the housekeeping cDNA clones irrelevant to the production of the protein of interest, leading to great reduction in the number of cDNA clones for screening. Methods other than differential cloning can be used for that purpose.
C) Third Step
Plasmids are prepared from the above-sorted cDNA clones the xcex phage vector of the invention and transfected into a host cells capable of producing the protein of interest. The host cells producing the protein of interest are identified by screening assay for the activity or function or with a specific antibody.
Specific examples of the host cells capable of producing the protein of interest include, but are not limited to, COS-1 cells.
D) Fourth Step
The plasmid is prepared from host cells such as E. coli, and then a gene of interest is isolated from the plasmid.